This invention relates to the acquired immune deficiency syndrome (AIDS) and, in particular, to assays which function as prognostic indicators of the development of AIDS by people infected with the human immunodeficiency virus (HIV).
The human immunodeficiency virus has been established as the etiologic agent of AIDS, chronically infecting and eventually killing cells critical to normal immunologic function. In addition to producing end-stage AIDS, HIV directly or indirectly contributes to immunologic dysfunction characterized clinically by the development of persistent generalized lymphadenopathy and AIDS-related complex (ARC). HIV-infected patients are usually staged by clinical findings and the numbers of CD4 positive lymphocytes (helper cells).
Patients infected with HIV can remain essentially symptom free for years, e.g., three years or longer, and not all HIV-infected people actually progress to AIDS. See, for example, Weber et al., "Human immunodeficiency virus infection in two cohorts of homosexual men: neutralizing sera and association of anti-gag antibody with prognosis," Lancet, Jan. 17, 1987, pages 119-121. As a result, extensive efforts have been made to develop laboratory tests which are prognostic indicators of the development of AIDS, ARC and other AIDS-related conditions by persons infected with HIV. See, for example, Spira, et al., "Human Immunodeficiency Virus Viremia as a Prognostic Indicator in Homosexual Men with Lymphadenopathy Syndrome," The New England Journal of Medicine, Vol. 317, No. 17, Oct. 22, 1987, pages 1093-1094. The need for predictive tests has become increasingly important as anti-HIV therapies directed at suppressing HIV replication have been developed. See, for example, Forster et al., "Decline of Anti-p24 Antibody Precedes Antigenaemia as Correlate of Prognosis in HIV-1 Infection," AIDS, 1987, Vol. 1, No. 4, pages 235-240.
One indicator which has been considered is the level of p24 antigen in a patient's serum, i.e., p24 antigenemia. See, for example, Wolf, et al., "Risk of AIDS Related Complex and AIDS in Homosexual Men with Persistent HIV Antigenaemia," British Medical Journal, Vol. 295, 1987, pages 569-572. In particular, an increase in the level of p24 antigen has been associated with progression to AIDS. Thus, Forster et al., supra, report that in 40% of the AIDS patients in their study, detectable levels of p24 antigen were seen 2 years prior to the actual development of AIDS.
The level of anti-p24 antibodies in a patient's serum has also been considered for use as a prognostic indicator. In this case, a decrease in the level of anti-p24 antibodies has been associated with progression to AIDS. See Biggar, et. al., "Variation in Human T Lymphotropic Virus III (HTLV-III) Antibodies in Homosexual Men: Decline Before Onset of Illness Related to Acquired Immune Deficiency Syndrome (AIDS)," British Medical Journal, Volume 291, Oct. 12, 1985, pages 997-998; Lange, et al., "Distinct IgG Recognition Patterns During Progression of Subclinical and Clinical Infection with Lymphadenopathy Associated Virus/Human T Lymphotropic Virus," British Medical Journal, Vol. 292, Jan. 25, 1986, pages 228-230; Pan et. al., "Patterns of Antibody Response in Individuals Infected with the Human Immunodeficiency Virus," Journal of Infectious Diseases, Vol. 155, No. 4, Apr. 1987, pages 626-632: Weber, et al., supra. As reported by Forster et al., supra, this indicator can provide predictive information up to 18 months earlier than the antigenemia indicator. (Note that Groopman, et al., "Serological Characterization of HTLV-III Infection in AIDS and Related Disorders," Journal of Infectious Diseases, Vol. 153, No. 4, Apr. 1986, pages 736-742, have found anti-p24 antibodies in both symptomatic and asymptomatic seropositive individuals.)
A number of recent studies have concluded that the combination of an increasing p24 antigen level and a declining anti-p24 antibody level is predictive of HIV-related clinical complications, including AIDS. See Allain, et al., "Long-Term Evaluation of HIV Antigen and Antibodies to p24 and gp41 in Patients with Hemophilia," The New England Journal of Medicine, Vol. 317, No. 18, Oct. 29, 1987, pages 1114-1121: Forster, et al., supra: Goudsmit, et al., "Antigenemia and Antibody Titers to Core and Envelope Antigens in AIDS, AIDS-Related Complex, and Subclinical Human Immunodeficiency Virus Infection," The Journal of Infectious Diseases, Vol. 155, No. 3, Mar. 1987, pages 558-560; Pedersen, et al., "Temporal Relation of Antigenaemia and Loss of Antibodies to Core Antigens to Development of Clinical Disease in HIV Infection," Britisn Medical Journal, Vol. 295, 1987, pages 567-569.
Various highly-sensitive assays for p24 antigen levels have been developed and are available from such companies as Abbott Laboratories (North Chicago, Ill.) and E. I. Du Pont de Nemours & Co. (Biomedical Products Department, Boston, Mass.). See, for example, Allain et al., supra: Forster, et al., supra; Goudsmit, et al., supra; Wolf, et al., supra. These assays employ a sandwich assay format in which serum samples are incubated with bound and enzyme-labelled anti-p24 antibodies to form an antibody/p24-antigen/antibody sandwich. Concentrations of p24 antigen are determined from standard curves obtained by performing the assay on a series of standards containing known amounts of p24 antigen. With these assays, p24 antigen levels on the order of 50 picograms/milliliter can be detected. See also Pedersen, et al., supra.
Assays for anti-p24 antibodies have also been developed. These assays suffer from a number of problems. The early assays, which continue to be used, employed Western blot techniques in which HIV virus proteins were separated by SDS-PAGE, transferred to a nitrocellulose sheet, incubated with the test serum, and developed using an antihuman IgG from an appropriate animal and a suitable enzyme-labeling system. See Biggar, et al., supra: Lange, et al., supra: Groopman, et al., supra: Pan, et al., supra. These assays were obviously difficult to perform, time consuming, and not well-suited for use in a clinical laboratory setting. See Pan, et al., supra. Also, the assays did not give reliable quantitative measures of anti-p24 antibodies in sera being tested. Radioimmunoprecipitation assays (RIPA) using radiolabeled antigens, immobilized test sera, and SDS-PAGE separation, have also been employed. See, for example, Weber, et al., supra. These assays suffer from the same problems as the Western blot assays and are not suitable for wide spread clinical use.
More recently, enzyme immunoassays for anti-p24 antibodies have been developed. See, for example, Forster, et al., supra. In particular, Abbott Laboratories has developed an assay in which beads coated with recombinant HIV core antigen, including p24 antigen, are incubated with serial dilutions of the serum being analyzed and with enzyme-labelled antibody to HIV core antigen. After a 16-22 hour incubation, the beads are washed and developed using a suitable substrate for the enzyme label. Since this is a competitive assay, the amount of antibody in the sample is inversely related to the intensity of the color generated during the development step. Positive and negative controls are run to establish a cutoff level for the presence of anti-p24 antibody, i.e., the midpoint between the controls. Titers are defined as the highest dilution which produces a color level below the cutoff. See Goudsmit, et al., supra; Pedersen, et al., supra. A similar competitive assay has been described by Weber, et al., supra. Significantly, Weber, et al., found that the classical radioimmunoprecipitation procedure was more sensitive than the competitive assay procedure. In addition to these competitive assays, direct ELISA assays for anti-p24 antibodies employing immobilized antigen and enzyme-labelled anti-human globulin antibodies have also been considered.
The primary disadvantage of these assay systems is that they employ serial dilutions of the patient's serum. Accordingly, in essence, numerous individual assays and measurements must be made to arrive at an antibody titer. This makes the assays difficult to use, labor intensive, and expensive. Indeed, Pedersen, et al., supra, after specifically pointing out that prognostic tests must be simple to do and interpret, recommended using a p24 antigen test for selecting patients for antiviral treatment, but significantly did not recommend using the Abbott competitive enzyme immunoassay for anti-p24 antibodies for this purpose.
Recently, a variation of the Abbott assay has been reported by Allain, et al., supra, in which titers are calculated by comparison with a reference curve obtained by serial dilutions of a sample containing standard antibody. The range of titers which can be measured by the modified assay is only between 1:2 and 1:256, and the assay requires the determination of a cutoff point for each series of assays so that the recorded optical densities can be transformed to titers using the standard curve.
Significantly, with regard to the enzyme immunoassays for anti-p24 antibodies, as well as all of the other assays for these antibodies, there is no integration between the assay for antibody levels and an assay for p24 antigen levels. As discussed above, it is the combination of a falling antibody level and a rising antigen level which appears to be most predictive of the clinical course of an HIV infection. Yet, as presently practiced, completely separate assays are being performed for these two important prognostic indicators.